Viral fusogens are dynamic proteins that undergo large rearrangements to facilitate the merger of viral and target cell membranes. In Herpes simplex viruses (HSV-1 and HSV-2), this fusion event results from the sequential action of four viral glycoproteins, gD, gH/gL, and the fusogen gB. HSV causes lifelong, latent infections that reactivate to cause ailments ranging from cold sores to blindness and encephalitis, and the knowledge of how gB mediates membrane fusion during viral entry may suggest approaches for blocking entry, thereby preventing infections in the first place. The fusogenic mechanism of gB remains unclear, but it is likely to be unique from that of other fusogens due to the unusual reliance of gB upon other viral glycoproteins. A major barrier to elucidating the HSV-specific refolding process and its regulation is that while the structure of the inactive, postfusion form o the gB ectodomain is available, the structure of active, prefusion gB remains elusive. Furthermore, important membrane-interacting regulatory regions of gB, its membrane proximal region (MPR), transmembrane domain (TMD), and cytoplasmic domain (cytodomain), have not yet been resolved. The goals of this application are to determine the structure of full-length postfusion gB and characterize prefusion gB, requisite steps towards unraveling the HSV fusion mechanism. The prefusion form of gB has been the target of several prior investigations, but all of these studies focused on its soluble ectodomain, a typical approach to the study of fusogens. In contrast, this proposal is based upon the hypothesis that gB is maintained in its metastable prefusion conformation through the interaction of its MPR, TMD, and cytodomain with the viral membrane. In essence, the contacts that gB makes with the membrane are critical to its structure and function. Three specific aims underpinned by this hypothesis will be used to study the pre and postfusion gB structures. First, full-length HSV-1 gB will be crystallized in detergent or a detergent/lipid mixture to determine its postfusion structure and reveal the architecture of the membrane proximal region, transmembrane domain, and cytodomain. Information on the interaction of the cytodomain with the membrane will also be obtained by electron spin resonance (ESR) spectroscopy. Second, prefusion gB will be captured in lipid bilayer disks and characterized in this membrane environment by electron microscopy and ESR. Finally, Frster resonance energy transfer (FRET) will be used to assess large conformational changes that are predicted for the prefusion to postfusion transition. Together, these experiments are expected to reveal long- sought details about the prefusion structure and the conformational changes that gB undergoes, illuminating the complex herpesvirus entry process and advancing our general knowledge of how fusogens work.